This invention relates to macromolecular conjugates, such as nucleic acid hybridization probes and immunological probes, useful as research and diagnostic tools. More particularly, it relates to making macromolecular conjugates by employing a universal conjugating agent.
Probe technology is emerging as a powerful tool in diagnostic testing, detection of genetic defects and the mapping of prokaryotic and eukaryotic genomes. Nucleic acids are characteristic of and therefore may be used to indicate the presence of a particular genus or species of bacteria or type of virus. They may indicate the presence of genes for pathogenicity or for antibiotic resistance or for a particular genetic disease.
Nucleic acid hybridization probes are well known tools of molecular biology. Grunstein et al., Proc. Natl. Acad. Sci. USA 72:3961 (1975) and Southern, J. Mol. Biol. 98:503 (1975), describe hybridization techniques using radiolabeled nucleic acid probes.
Diagnostic tests based on culture techniques are often difficult and slow to produce results. Many pathogens, including viruses and bacteria, require incubation from overnight to six weeks to yield diagnostic results. In addition, some organisms cannot be cultured.
Monoclonal antibody techniques often have limited specificity. They are subject to undesirable cross reactions and the inability to detect antigenic variants. Nucleic acid hybridization probes have the advantages over other methods of speed and high specificity. However, existing hybridization techniques which utilize radioisotopes introduce additional expenses in disposal of radioactive waste products and monitoring personnel and the work place for contamination. Autoradiographic detection may require up to two weeks of exposure. These techniques are not suited to commercial areas such as clinical diagnosis.
One technique under development to overcome the drawbacks inherent in radioisotopic probes is the non-radioactive labeling of nucleic acids. Any labeling group that does not prevent hybridization of the nucleic acid with its target may be used to form a probe. After hybridization with a target nucleic acid, the labeled duplex is reacted with additional reagents to provide a signal. The most common method of indirect labeling is to attach biotin, a small vitamin, to the nucleic acid, using chemical or enzymatic techniques. Following hybridization, the biotin is detected with avidin, an egg white protein. Avidin may be labeled with an enzyme or a fluorochrome. Enzyme labels are detected by a calorimetric reaction. Fluorochrome labels are detected by illumination with light of a specific wavelength.
The ability of biotin to bind to avidin has been exploited in hybridization assays. U.S. Pat. No.4,581,333 to Kourilsky, et al., for example, discloses the use of avidin-bound enzymes to detect hybridization between biotinylated DNA probes and a particular nucleic acid molecule of interest. European Patent Application 133,473 discloses the use of biotin-avidin bridging agents as well as sugar-binding lectins to link the proteins and nucleic acid molecules used in a hybridization assay.
Avidin-biotin techniques are complex because of the many steps involved: probe is labeled with biotin, avidin is labeled with an enzyme or fluorochrome, the biotinylated probe is, hybridized to the target, and the labeled avidin is reacted with the biotinylated probe, the labeled avidin is detected by a colorimetric reaction or by illumination with light. A method of attaching a signal-generating enzyme or fluorochrome directly to the DNA to make a probe would be advantageous because the diagnostic test would be greatly simplified: the DNA would be labeled with the enzyme or fluorochrome, hybridized to the target, and detected by a colorimetric reaction or by illumination with light.
Antibodies have been employed as bifunctional conjugating agents to link proteins to nucleic acid molecules. U.S. Pat. No. 4,556,643 to Paau et al. discloses the use of antibodies, as well as DNA binding proteins, in hybridization assays. European Patent Application 146,815 discloses hybridization assays which employ antibodies capable of binding to a DNA intercalator molecule. Similar inventions are disclosed in U.S. Pat. No. 4,582,789 to Sheldon and in European Patent Application 131,830. These methods also require complex assay systems as compared to a directly labeled DNA probe.
A large number of covalent conjugating agents are known in the immunoassay art, where they have been used to directly attach such labels to antibodies or antigens. Antibodies or antigens labeled in this manner are sometimes referred to as immunologized probes. Such agents are also often used to attach immunogens to carriers. U.S. Pat. No. 4,469,797 to Albarella, discloses digoxigenin derivatives capable of acting as bifunctional coupling agents to link immunoglobulins to polypeptide carriers. U.S. Pat. No. 4,378,428 to Farina et al., discloses covalent conjugating agents which may be used in a homogeneous immunoassay. U.S. Pat. No. 4,302,534 to Halmann, et al., discloses the use of either antigen or antibody labeled with peroxidase in an immunoassay. U.S. Pat. No. 3,951,748 to Devlin, discloses an immunoassay in which a coupling agent immobilizes protein molecules to an insoluble matrix. U.S. Pat. No. 3,817,837 to Rubenstein discloses an immunoassay which employs a covalent conjugating agent to bind an enzyme ligand. The use of biotin as a bifunctional conjugating agent is disclosed in U.S. Pat. No. 4,298,685 to Parikh et al. U.S. Pat. No. 4,241,177 to Singh, additionally discloses similar covalent conjugating agents.
A small number of proteins bind readily to deoxyribonucleic acid (DNA). These are referred to as DNA binding proteins. Known DNA-binding proteins such as histones, RecA and single-stranded DNA binding protein (SSB) have been employed in hybridization and diagnostic assays. European Patent Applications 183,822 and 164,876 disclose hybridization methods for identifying known genetic sequences in a target DNA molecule which employ DNA binding proteins such as E. coli RecA and SSB. Japanese Patent Application 56001351 discloses a method for quantitatively analyzing DNA binding protein by affinity chromatography using a carrier to which DNA is linked. Histones and some other positively charged proteins form excellent DNA binding proteins. These proteins are candidates for labeling groups. However, most proteins that are signal generating or that would react with signal molecules are not DNA binding proteins. These may be attached to nucleic acids by covalent conjugating agents. Conjugate probes are made by bonding a labeling group to a nucleic acid by using a conjugating agent.
A number of methods are known for covalently crosslinking proteins to nucleic acid molecules. European Patent Application 151,001 discloses a polynucleotide which is covalently crosslinked to a protein molecule. European Patent Application EP 138,357 discloses additional bifunctional covalent conjugating agents. U.S. Pat. No. 4,587,044 to Miller et al. discloses nucleic acids which are modified by esterification with a saturated or unsaturated aliphatic dicarboxylic acid or anhydride to produce a molecule capable of being crosslinked to a protein. U.S. Pat. No. 4,699,876 to Libeskind, discloses a number of bifunctional cross-linking agents including N-succinimidyl 4-glyoxalylbenzoate, carbonyl imidazole, dimethyl superimidate, 1-ethyl,3-dimethylaminopropylcarbodiimide, paranitropenyl 3(2-bromo,3-ketobutylsulfonyl)propionate or other active esters, glutaraldehyde and other suitable equivalent.
The use of glutaraldehyde as a covalent conjugating agent capable of binding nondetectable proteins to nucleic acid molecules is disclosed by Borel et al., J. Immunol. Met. 67:289-302 (1984). Borel et al. describe a 2 stage process in which an oligonucleotide is incubated with glutaraldehyde and the oligonucleotide-glutaraldehyde conjugate is then incubated with the desired protein to produce the oligonucleotide-protein conjugate. Glutaraldehyde has also been used to covalently attach histones to nucleic acids, as reported by Renz, M., Eur. Mol. Biol. Organ. J. 2:817 (1983). Histones are lysine rich (i.e. positively charged) DNA-binding proteins. Glutaraldehyde adds covalent bonds to these two moieties that are already matched by a natural strong affinity. The tight binding between nucleic acids and lysine rich histones allows efficient glutaraldehyde crosslinking at low concentrations.
It is not always possible to find proteins that are easily detectable and also have a strong natural affinity for nucleic acids. Proteins that do not have a natural affinity for nucleic acids will not allow the same efficient glutaraldehyde crosslinking. Therefore, one would expect varying degrees of success from attempts to crosslink a variety of proteins to nucleic acids using glutaraldehyde or any other conjugating agent.
Renz et al., Nucleic Acid Res. 12:3435-3444 (1984) considered ionic binding between the protein and the nucleic acid to be essential to the success of a conjugating agent which covalently binds them. To convert horseradish peroxidase into a DNA-binding protein, polyethylenimine carrying primary amino groups was fused to horseradish peroxidase with p-benzoquinone as the crosslinking agent. This modified horseradish peroxidase had an increased affinity for single stranded DNA and could be covalently conjugated with glutaraldehyde.
U.S. Pat. No. 4,166,105 to Hirschfield discusses linking a labeling group to an antibody using non-polymerized polyaldehydes such as glutaraldehyde.
U.S. Pat. No. 4,267,234 to Rembaum links biological molecules to water insoluble polyglutaraldehyde microspheres, to produce water insolubilized biomolecules.
A wide variety of cationic detergents possessing hydrophobic groups on one end and positively-charged groups on the other may be prepared. Such detergents are disclosed by, for example, U.S. Pat. No. 4,235,759 to Ohbu and U.S. Pat. No. 4,454,060 to Lai, et al. Such detergents have found use as cleaning agents such as shampoos, bubble baths and skin cleansers, etc.
Detergents were found to be DNA-protein conjugating agents, as disclosed by U.S. Pat. No. 4,873,187 to Taub.
Place et al., J. Immunol. Met. 48:251-260 (1982) discloses a method of improving the adsorption of protein to plastic surfaces to improve the reliability of quantitative solid phase immunoassays. Place et al. observed that antibody to hepatitis B surface antigen (anti-HBSAg) could be partially removed from a plastic surface by serum albumin. Removal could be substantially prevented if the wells of a polyvinyl chloride (PVC) microtiter plate were first treated with 2% polyglutaraldehyde. Monomeric glutaraldehyde was, in contrast, ineffective to improve the binding of protein to plastic.
It is therefore an object of the present invention to provide a new method of forming a conjugate probe formed between a macromolecule and a labeling group.
It is a further object of the present invention to provide a conjugate probe made from any macromolecule by binding to any labeling group.
It is another object of the present invention to provide hybrids and a method of making hybrids between the conjugate probe and its target nucleic acid.
It is yet another object of the present invention to provide a method of detecting the presence of a target nucleic acid by detecting the presence of a labeling group that is bound to a hybrid.
It is another object of the present invention to provide test kits for making macromolecular conjugates and for performing hybridization testing using the conjugate probe and a hybridization medium.